Triple Thorpe-Ingold Effect in the Synthesis of 18-Membered C3 Symmetric Lactams Stacking as Endless Supramolecular Tubes.

Three C3 symmetric macrolactams were very efficiently cyclized from their linear precursors. Adequately located substituents are responsible for the enhancement of reactivity that is not observed in the unsubstituted parent. DFT calculations show that the properly folded cyclization precursor, the reactive conformer, is more populated than other conformers, leading to a decrease of free energy of activation. The crystal structure of the ring substituted with three very bulky esters indicates that tubular stacking is preserved.

Second Harmonic Generation Signatures of Supramolecular Assemblies Based on Amide Moieties.

Targeting the use of the second harmonic generation (SHG) as a bioimaging technique to unravel the formation of aggregates, the SHG first hyperpolarizabilities ( ß ${\beta }$ ) of assemblies of benzene-1,3,5-tricarboxamide derivatives have been evaluated at the density functional theory level. Calculations have revealed that i) the assemblies exhibit SHG responses and the total first hyperpolarizability responses of the aggregates are evolving with their size. The largest aggregation effect is a 18-times increase for ß H R S ${{\beta }_{HRS}}$ of B4 when going from the monomer to the pentamer, that ii) the intrinsic SHG responses described by the hyper-Rayleigh Scattering ß ${\beta }$ are enhanced in presence of iodine atoms on the phenyl core, that iii) the side chains affect the relative orientation of the dipole moment and first hyperpolarizability vectors, which impacts more the EFISHG quantities than their moduli, and that iv) the radial component to ß ${\beta }$ is dominant for the compounds having the largest responses. These results have been obtained using the sequential molecular dynamics then quantum mechanics approach to account for dynamic structural effects on the SHG responses.

Enhancing the Drug-Like Profile of a Potent Peptide PACE4 Inhibitor by the Formation of a Host-Guest Inclusion Complex with ß-Cyclodextrin.

The enzyme PACE4 has been validated as a promising therapeutic target to expand the range of prostate cancer (PCa) treatments. In recent years, we have developed a potent peptidomimetic inhibitor, namely, compound C23 (Ac-(DLeu)LLLRVK-4-amidinobenzylamide). Like many peptides, C23 suffers from an unfavorable drug-like profile which, despite our efforts, has not yet benefited from the usual SAR studies. Hence, we turned our attention toward a novel formulation strategy, i.e., the use of cyclodextrins (CDs). CDs can benefit compounds through the formation of "host-guest" complexes, shielding the guest from degradation and enhancing biological survival. In this study, a series of ßCD-C23 complexes have been generated and their properties evaluated, including potency toward the enzyme in vitro, a cell-based proliferation assay, and stability in plasma. As a result, a new ßCD-formulated lead compound has been identified, which, in addition to being more soluble and more potent, also showed an improved stability profile.

Computational prediction of the supramolecular self-assembling properties of organic molecules: the role of conformational flexibility of amide moieties.

Two families of organic molecules with different backbones have been considered. The first family is based on a macrolactam-like unit that is constrained in a particular conformation. The second family is composed by a substituted central phenyl that allows a larger mobility for its substituents. They have however a common feature, three amide moieties (within the cycle for the macrolactam-like molecule and as substituents for the phenyl) that permit hydrogen bonding when molecules are stacked. In this study we propose a computational protocol to unravel the ability of the different families to self-assemble into organic nanotubes. Starting from the monomer and going towards larger assemblies like dimers, trimers, and pentamers we applied the different protocols to rationalize the behavior of the different assemblies. Both structures and thermodynamics were investigated to give a complete picture of the process. Thanks to the combination of a quantum mechanics approach and molecular dynamics simulations along with the use of tailored tools (non covalent interaction visualization) and techniques (umbrella sampling), we have been able to differentiate the two families and highlight the best candidate for self-assembling purposes.

Self-Assembly of C3 Symmetric Rigid Macrolactams into Very Polar and Porous Trigonal Crystals.

Cyclohexane and cyclotri-ß-alanyl have been used as scaffolds for the design of new C3 -symmetric rings incorporating conjugated alkenes and dienes. All three C3 -symmetric lactams share the same triangular shape and their crystal system is trigonal. They all belong to the R3 space group, R3m, R3 and R3c, for the increasingly large 12-, 18- and 24-membered rigid rings, respectively. All lactams stack on top of each other, through H-bonds and van der Waals noncovalent interactions, leading to endless supramolecular cylinders and tubes. The largest member of the family leads to tubes, the central pores of which is wide enough to let water in. A common feature of all the lactams is their very large dipole, of around 9 D, according to DFT calculations. Surprisingly, all the resulting cylinders and tubes pack side by side in the crystals, with all the dipoles pointing to the same direction. As a result, all three crystals are anisotropic and appear to be the first members of a new kind of highly polar crystals.

The Proprotein Convertase Subtilisin/Kexin Type 9-resistant R410S Low Density Lipoprotein Receptor Mutation: A NOVEL MECHANISM CAUSING FAMILIAL HYPERCHOLESTEROLEMIA.

Familial hypercholesterolemia (FH) is characterized by severely elevated low density lipoprotein (LDL) cholesterol. Herein, we identified an FH patient presenting novel compound heterozygote mutations R410S and G592E of the LDL receptor (LDLR). The patient responded modestly to maximum rosuvastatin plus ezetimibe therapy, even in combination with a PCSK9 monoclonal antibody injection. Using cell biology and molecular dynamics simulations, we aimed to define the underlying mechanism(s) by which these LDLR mutations affect LDL metabolism and lead to hypercholesterolemia. Our data showed that the LDLR-G592E is a class 2b mutant, because it mostly failed to exit the endoplasmic reticulum and was degraded. Even though LDLR-R410S and LDLR-WT were similar in levels of cell surface and total receptor and bound equally well to LDL or extracellular PCSK9, the LDLR-R410S was resistant to exogenous PCSK9-mediated degradation in endosomes/lysosomes and showed reduced LDL internalization and degradation relative to LDLR-WT. Evidence is provided for a tighter association of LDL with LDLR-R410S at acidic pH, a reduced LDL delivery to late endosomes/lysosomes, and an increased release in the medium of the bound/internalized LDL, as compared with LDLR-WT. These data suggested that LDLR-R410S recycles loaded with its LDL-cargo. Our findings demonstrate that LDLR-R410S represents an LDLR loss-of-function through a novel class 8 FH-causing mechanism, thereby rationalizing the observed phenotype.

Systematic replacement of amides by 1,4-disubstituted[1,2,3]triazoles in Leu-enkephalin and the impact on the delta opioid receptor activity.

Using Cu(I)-catalyzed azide-alkyne cycloaddition in a mixed classical organic phase and solid phase peptide synthesis approach, we synthesized four analogs of Leu-enkephalin to systematically replace amides by 1,4-disubstituted[1,2,3]triazoles. The peptidomimetics obtained were characterized by competitive binding, contractility assays and ERK1/2 phosphorylation. The present study reveals that the analog bearing a triazole between Phe and Leu retains some potency, more than all the others, suggesting that the hydrogen bond acceptor capacity of the last amide of Leu-enkephalin is essential for the biological activity of the peptide.

Simultaneous chirality transfer and structured aggregation of a cyclopeptide in a liquid crystal.

We report the finding that a chiral cyclopeptide dissolved in a nematic liquid crystal (LC) host could aggregate in a manner that is controlled by the texture (LC director configuration) of a cholesteric phase that is induced by the cyclopeptide itself. On one hand, with the fingerprint texture, where the helical axis formed by rotating LC molecules, that lies in the substrate plane, the cyclopeptide can use the LC texture as a template to aggregate and form long-range-ordered ribbons that mimic the helical configuration of the LC director. On the other hand, with the planar texture, where the helical axis is normal to the substrate plane, the cyclopeptide can migrate into the "oily-streak" defect regions of the cholesteric phase and stabilize a network of defects that dictates the electrooptical response of the LC. This is the first example of a molecular species exhibiting such a structured aggregation and defect stabilization effect in a cholesteric LC, but similar phenomena were previously reported for platinum nanoparticles and silica colloidal particles, respectively, dispersed in a cholesteric LC host. This study provides more evidence for the potential interest of exploring LCs as an anisotropic medium for mediating the aggregation and assembly of cyclopeptides.

Design and Structure-Activity Relationship of a Potent Furin Inhibitor Derived from Influenza Hemagglutinin.

Furin plays an important role in various pathological states, especially in bacterial and viral infections. A detailed understanding of the structural requirements for inhibitors targeting this enzyme is crucial to develop new therapeutic strategies in infectious diseases, including an urgent unmet need for SARS-CoV-2 infection. Previously, we have identified a potent furin inhibitor, peptide Ac-RARRRKKRT-NH 2 (CF1), based on the highly pathogenic avian influenza hemagglutinin. The goal of this study was to determine how its N-terminal part (the P8-P5 positions) affects its activity profile. To do so, the positional-scanning libraries of individual peptides modified at the selected positions with natural amino acids were generated. Subsequently, the best substitutions were combined together and/or replaced by unnatural residues to expand our investigations. The results reveal that the affinity of CF1 can be improved (2-2.5-fold) by substituting its P5 position with the small hydrophobic residues (Ile or Val) or a basic Lys.

Promising Performance of 4HMS, a New Zirconium-89 Octadendate Chelator.

Over the last decade, the interest in zirconium-89 (89Zr) as a positron-emitting radionuclide increased considerably because of its standardized production and its physical half-life (78.41 h), which matches the biological half-life of antibodies and its successful use in preclinical and clinical applications. So far, desferrioxamine (DFO), a commercially available chelator, has been mainly used as a bifunctional chelating system. However, there are some concerns regarding the in vivo stability of the [89Zr]Zr-DFO complex. In this study, we report the synthesis of an acyclic N-hydroxy-N-methyl succinamide-based chelator (4HMS) with 8 coordination sites and our first investigations into the use of this new chelator for 89Zr complexation. In vitro and in vivo comparative studies with [89Zr]Zr-4HMS and [89Zr]Zr-DFO are presented. The 4HMS chelator was synthesized in four steps starting with an excellent overall yield. Both chelators were quantitatively labeled with 89Zr within 5-10 min at pH 7 and room temperature; the molar activity of [89Zr]Zr-4HMS exceeded (>3 times) that of [89Zr]Zr-DFO. [89Zr]Zr-4HMS remained stable against transmetalation and transchelation and cleared from most tissues within 24 h. The kidney, liver, bone, and spleen uptakes were significantly low for this 89Zr-complex. Positron emission tomography images were in accordance with the results of the biodistribution in healthy mice. Based on DFT calculations, a rationale is provided for the high stability of 89Zr-4HMS. This makes 4HMS a promising chelator for future development of 89Zr-radiopharmaceuticals.

Cyclic peptide-polymer complexes and their self-assembly.

The efficient synthesis of novel chiral cyclic peptides cyclo[NHCHX-CH=CHCH(2)CO(NHCH(2)CH=CHCH(2)CO)(2)] designed to develop hydrogen-bonding interactions with suitable polymers is described. Complexation of a carboxylic acid derivatized cyclic peptide 2 (X = CH(2)OCOCH(2)CH(2)CO(2)H) capable of self-assembling as "endless" tubes, with poly(vinyl alcohol) (PVA) led to a vast weak-interaction network, in which the cyclopeptide developed extensive hydrogen-bonding interactions with the hydroxyl groups of PVA through not only the carboxylic acid, but also its ester carbonyl and amide groups. In aqueous solution, the peptide/PVA complexes self-assemble into long-grain ricelike aggregates compatible with the stacking of cyclic peptides through intercycle hydrogen bonds. Upon casting on silicon wafer, the anisotropic aggregates can coalesce to form filaments tens of micrometers long. The study demonstrates that complexing functionalized cyclic peptides with polymers through hydrogen bonding is a useful approach for using polymers to mediate the self-assembly and self-organization of cyclic peptides.

Efficient synthesis of nevirapine analogs to study its metabolic profile by click fishing.

Knowledge of the biotransformation and pharmacokinetics of the antiretroviral agent nevirapine is still insufficient. In order to trace rash inducing metabolites of nevirapine, we devised a short and efficient multi-gram synthesis of a nevirapine analog that can be coupled to azide containing compounds by click chemistry.

Enhanced anti-tumor activity of the Multi-Leu peptide PACE4 inhibitor transformed into an albumin-bound tumor-targeting prodrug.

The proprotein convertase PACE4 has been validated as a potential target to develop new therapeutic interventions in prostate cancer (PCa). So far, the most effective compound blocking the activity of this enzyme has been designed based on the structure of a small peptide Ac-LLLLRVKR-NH2 known as the Multi-Leu (ML) peptide. Optimization of this scaffold led to the synthesis of compound C23 (Ac-[DLeu]LLLRVK-amidinobenzylamide) with a potent in vivo inhibitory effect on the tumor growth. However, further developments of PACE4 inhibitors may require additional improvements to counter their rapid renal clearance and to increase their tumor targeting efficiency. Herein, we explored the transformation of the ML-peptide into an albumin-binding prodrug containing a tumor specific release mechanism based on the prostate-specific antigen. Our data confirms that intravenous treatment using the ML-peptide alone has little effect on tumor growth, whereas by using the ML-prodrug in LNCaP xenograft-bearing mice it was significantly reduced. Additionally, excellent in vivo stability and tumor-targeting efficiency was demonstrated using a radiolabelled version of this compound. Taken together, these results provide a solid foundation for further development of targeted PACE4 inhibition in PCa.

N-Guanidyl and C-Tetrazole Leu-Enkephalin Derivatives: Efficient Mu and Delta Opioid Receptor Agonists with Improved Pharmacological Properties.

Leu-enkephalin and d-Ala2-Leu-enkephalin were modified at their N- and C-termini with guanidyl and tetrazole groups. The resulting molecules were prepared in solution or by solid phase peptide synthesis. The affinity of the different analogues at mu (MOP) and delta opioid receptors (DOP) was then assessed by competitive binding in stably transfected DOP and MOP HEK293 cells. Inhibition of cAMP production and recruitment of ß-arrestin were also investigated. Finally, lipophilicity (logD7.4) and plasma stability of each compound were measured. Compared to the native ligands, we found that the replacement of the terminal carboxylate by a tetrazole slightly decreased both the affinity at mu and delta opioid receptors as well as the half-life. By contrast, replacing the ammonium at the N-terminus with a guanidyl significantly improved the affinity, the potency, as well as the lipophilicity and the stability of the resulting peptides. Replacing the glycine residue with a d-alanine in position 2 consistently improved the potency as well as the stability of the analogues. The best peptidomimetic of the whole series, guanidyl-Tyr-d-Ala-Gly-Phe-Leu-tetrazole, displayed sub-nanomolar affinity and an increased lipophilicity. Moreover, it proved to be stable in plasma for up to 24 h, suggesting that the modifications are protecting the compound against protease degradation.

Rational design and gas-phase characterization of molecular capsules by self-assembly of a symmetric hexasubstituted benzene with seven-membered lactams.

A novel very simple C3-symmetric lactam has been rationally designed to self-assemble as dimers or larger platonic solid capsules. Its core flat benzene ring bears three seven-membered lactams, resulting in tripod molecules that aggregate into robust tetrameric capsules. The self-assembly process was templated by tetraethylammonium cations and proven to be reversible by ESI spectroscopy in various solvents.

Efficient parallel synthesis of macrocyclic peptidomimetics.

A new method for solid phase parallel synthesis of chemically and conformationally diverse macrocyclic peptidomimetics is reported. A key feature of the method is access to broad chemical and conformational diversity. Synthesis and mechanistic studies on the macrocyclization step are reported.

Increasing C-Terminal Hydrophobicity Improves the Cell Permeability and Antiproliferative Activity of PACE4 Inhibitors against Prostate Cancer Cell Lines.

The serine protease, PACE4, is a proprotein convertase that plays a substantial role in malignancy of prostate cancer. Our initial selective PACE4 inhibitor (Ac-LLLLRVKR-NH2) has evolved to the current lead compound C23 (Ac-dLeu-LLLRVK-Amba), which is active both in vitro and in vivo. By screening natural residues, except Cys, in C-terminal P1' position, it was established that increasing hydrophobicity was improving cell permeability, which was directly translated into PCa cells antiproliferative activity. This cell antiproliferation enhancement seems independent from effect of P1' residue on PACE4 affinity. Replacement of P1-Amba of C23 by Acpa (( S)-2-amino-3-(4-carbamimidoylphenyl)propanoic acid) followed by addition of tryptamine in P1' resulted in compound 32 exhibiting superior PCa cells antiproliferative activity over the reference compound C23 (3-fold). This study sheds light on key factors that improve cell penetrating property and antiproliferative activity of PACE4 inhibitors.

Improving the Selectivity of PACE4 Inhibitors through Modifications of the P1 Residue.

Paired basic amino acid cleaving enzyme 4 (PACE4), a serine endoprotease of the proprotein convertases family, has been recognized as a promising target for prostate cancer. We previously reported a selective and potent peptide-based inhibitor for PACE4, named the multi-Leu peptide (Ac-LLLLRVKR-NH2 sequence), which was then modified into a more potent and stable compound named C23 with the following structure: Ac-dLeu-LLLRVK-Amba (Amba: 4-amidinobenzylamide). Despite improvements in both in vitro and in vivo profiles of C23, its selectivity for PACE4 over furin was significantly reduced. We examined other Arg-mimetics instead of Amba to regain the lost selectivity. Our results indicated that the replacement of Amba with 5-(aminomethyl)picolinimidamide increased affinity for PACE4 and restored selectivity. Our results also provide a better insight on how structural differences between S1 pockets of PACE4 and furin could be employed in the rational design of selective inhibitors.

Diffusion of chiral molecules and propagation of structural chirality in anisotropic liquids.

Diffusion in nature is usually considered as a smooth redistribution process. However, it appears that the diffusion of chiral molecules and the propagation of chirality may proceed in quite different ways. Indeed, in the present work, unexpected quantization of the spatial concentration of chiral molecules is discovered in self-aligned molecular liquids. It is shown that the interpenetration of two liquids is forming discrete diffusion barrier walls resulting in steplike concentration distribution of chiral molecules in space. The concentration gradient is at least an order of magnitude stronger from both sides of the barrier wall compared to the gradient between those walls. It is also shown that this microscopic diffusion process may be controlled by macroscopic boundary conditions imposed on the host molecular system. Both of those phenomena are related to the collective long-range orientational "elastic" interactions of molecules of the host. The observed phenomena may radically change our understanding of diffusion of chiral molecules, among others, in biological tissue, which contains many examples of self-aligned molecular liquids. This, in turn, has the potential to revolutionize drug design and delivery techniques.

Rational Design of a Highly Potent and Selective Peptide Inhibitor of PACE4 by Salt Bridge Interaction with D160 at Position P3.

PACE4, a member of the proprotein convertases (PCs) family of serine proteases, is a validated target for prostate cancer. Our group has developed a potent and selective PACE4 inhibitor: Ac-LLLLRVKR-NH2 . In seeking for modifications to increase the selectivity of this ligand toward PACE4, we replaced one of its P3 Val methyl groups with a basic group capable of forming a salt bridge with D160 of PACE4. The resulting inhibitor is eight times more potent than the P3 Val parent inhibitor and two times more selective over furin, because the equivalent salt bridge with furin E257 is not optimal. Moreover, the ß-branched nature of the new P3 residue favors the extended ß-sheet conformation usually associated with substrates of proteases. This work provides new insight for better understanding of ß-sheet backbone-backbone interactions between serine proteases and their peptidic ligands.